Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae

As a first step towards a vaccine against diarrhoeal disease caused by enterotoxigenic Escherichia coli (ETEC), we have studied the expression of several ETEC antigens in the live attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Colonization factors (CF) CFA/I, CS3, and CS6 were expressed at the surface of V. cholerae CVD 103-HgR. Both CFA/I and CS3 required the co-expression of a positive regulator for expression, while CS6 was expressed without regulation. Up-regulation of CF expression in V. cholerae was very efficient, so that high amounts of CFA/I and CS3 similar to those in wild-type ETEC were synthesized from chromosomally integrated CF and positive regulator loci. Increasing either the operon and/or the positive regulator gene dosage resulted in only a small increase in CFA/I and CS3 expression. In contrast, the level of expression of the non-regulated CS6 fimbriae appeared to be more dependent on gene dosage. While CF expression in wild-type ETEC is known to be tightly thermoregulated and medium dependent, it seems to be less stringent in V. cholerae. Finally, co-expression of two or three CFs in the same strain was efficient even under the control of one single regulator gene.

Leukocyte populations and mRNA expression of inflammatory factors in quarter milk fractions at different somatic cell score levels in dairy cows

The effect of somatic cell count (SCC) and milk fraction on milk composition, distribution of cell populations, and mRNA expression of various inflammatory parameters was studied. Therefore, quarter milk samples were defined as cisternal (C), first 400 g of alveolar (A1), and remaining alveolar milk (A2) during the course of milking. Quarters were assigned to 4 groups according to their total SCC: 1) <12 x 10(3)/mL, 2) 12 to 100 x 10(3)/mL, 3) 100 to 350 x 10(3)/mL, and 4) >350 x 10(3)/mL. Milk constituents of interest were SCC, fat, protein, lactose sodium, and chloride ions as well as electrical conductivity. Cell populations were classified into lymphocytes, macrophages, and neutrophils (PMN). The mRNA expression of the inflammatory factors tumor necrosis factor-alpha, interleukin-1beta, cyclooxygenase-2, lactoferrin, and lysozyme was measured via real-time, quantitative reverse transcription PCR. Somatic cell count decreased from highest levels in C to lowest levels in A1 and increased thereafter to A2 in all groups. Fat content increased from C to A2 and with increasing SCC level. Lactose decreased with increasing SCC level but remained unchanged during milking. Concentrations of sodium and chloride, and electrical conductivity increased with increasing SCC but were higher in C than in A1 and A2. Protein was not affected by milk fraction or SCC level. The distribution of leukocytes was dramatically influenced by milk fraction and SCC. Lymphocytes were the dominating cell population in group 1, but the proportion of lymphocytes was low in groups 2, 3, and 4. Macrophage proportion was highest in group 2 and decreased in groups 3 and 4, whereas that of PMN increased from group 2 to 4. The content of macrophages decreased during milking in all SCC groups whereas that of PMN increased. The proportion of lymphocytes was not affected by milk fraction. The mRNA expression of all inflammatory factors showed an increase with increasing SCC but minor changes occurred during milking. In conclusion, milk fraction and SCC level have a crucial influence on the distribution of leukocyte populations and several milk constituents. The surprisingly high content of lymphocytes and concomitantly low mRNA expression of inflammatory factors in quarters with SCC <12 x 10(3)/mL indicates a different and possibly reduced readiness of the immune system to respond to invading pathogens.

Progression of pulmonary hyperinflation and trapped gas associated with genetic and environmental factors in children with cystic fibrosis

BACKGROUND: Functional deterioration in cystic fibrosis (CF) may be reflected by increasing bronchial obstruction and, as recently shown, by ventilation inhomogeneities. This study investigated which physiological factors (airway obstruction, ventilation inhomogeneities, pulmonary hyperinflation, development of trapped gas) best express the decline in lung function, and what role specific CFTR genotypes and different types of bronchial infection may have upon this process. METHODS: Serial annual lung function tests, performed in 152 children (77 males; 75 females) with CF (age range: 6-18 y) provided data pertaining to functional residual capacity (FRCpleth, FRCMBNW), volume of trapped gas (VTG), effective specific airway resistance (sReff), lung clearance index (LCI), and forced expiratory indices (FVC, FEV1, FEF50). RESULTS: All lung function parameters showed progression with age. Pulmonary hyperinflation (FRCpleth > 2SDS) was already present in 39% of patients at age 6-8 yrs, increasing to 67% at age 18 yrs. The proportion of patients with VTG > 2SDS increased from 15% to 54% during this period. Children with severe pulmonary hyperinflation and trapped gas at age 6-8 yrs showed the most pronounced disease progression over time. Age related tracking of lung function parameters commences early in life, and is significantly influenced by specific CFTR genotypes. The group with chronic P. aeruginosa infection demonstrated most rapid progression in all lung function parameters, whilst those with chronic S. aureus infection had the slowest rate of progression. LCI, measured as an index of ventilation inhomogeneities was the most sensitive discriminator between the 3 types of infection examined (p < 0.0001). CONCLUSION: The relationships between lung function indices, CFTR genotypes and infective organisms observed in this study suggest that measurement of other lung function parameters, in addition to spirometry alone, may provide important information about disease progression in CF.

Protein buffering in model systems and in whole human saliva

The aim of this study was to quantify the buffer attributes (value, power, range and optimum) of two model systems for whole human resting saliva, the purified proteins from whole human resting saliva and single proteins. Two model systems, the first containing amyloglucosidase and lysozyme, and the second containing amyloglucosidase and alpha-amylase, were shown to provide, in combination with hydrogencarbonate and di-hydrogenphosphate, almost identical buffer attributes as whole human resting saliva. It was further demonstrated that changes in the protein concentration as small as 0.1% may change the buffer value of a buffer solution up to 15 times. Additionally, it was shown that there was a protein concentration change in the same range (0.16%) between saliva samples collected at the time periods of 13:00 and others collected at 9:00 am and 17:00. The mode of the protein expression changed between these samples corresponded to the change in basic buffer power and the change of the buffer value at pH 6.7. Finally, SDS Page and Ruthenium II tris (bathophenantroline disulfonate) staining unveiled a constant protein expression in all samples except for one 50 kDa protein band. As the change in the expression pattern of that 50 kDa protein band corresponded to the change in basic buffer power and the buffer value at pH 6.7, it was reasonable to conclude that this 50 kDa protein band may contain the protein(s) belonging to the protein buffer system of human saliva.

Insulin-like growth factors (IGFs), IGF binding proteins, and other endocrine factors in milk: role in the newborn

The role of colostrum and milk in the neonate has been chiefly recognized as a comprehensive nutrient foodstuff. In addition, the provision of colostrum-the first milk-for early immune capacity has been well documented for several species. Colostrum is additionally a rich and concentrated source of various factors that demonstrate biological activity in vitro. Three hypotheses have been proposed for the phenotypic function of these secreted bioactive components: (1) only mammary disposal, (2) mammary cell regulation, and (3) neonatal function [gastrointestinal tract (GIT) or systemic]. Traditionally, it was assumed that the development of the GIT is preprogrammed and not influenced by events occurring in the intestinal lumen. However, a large volume of research has demonstrated that colostrum (or milk-borne) bioactive components can basically contribute to the regulation of GIT growth and differentiation, while their role in postnatal development at physiological concentrations has remained elusive. Much of our current understanding is derived from cell culture and laboratory animals, but experimentation with agriculturally important species is taking place. This chapter provides an overview of work conducted primarily in neonatal calves and secondarily in other species on the effects on neonates of selected peptide endocrine factors (hormones, growth factors, in part cytokines) in colostrum. The primary focus will be on insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) and other bioactive peptides, but new interest and concern about steroids (especially estrogens) in milk are considered as well.

Comparative acute effects of the calcium channel blockers tiapamil, nisoldipine, and nifedipine on blood pressure and some regulatory factors in normal and hypertensive subjects

To clarify the pharmacological profile of the two new calcium channel blockers tiapamil and nisoldipine in humans, their acute effects as compared with those of the reference agent nifedipine were assessed in 10 normal subjects and 10 patients with essential hypertension. Blood pressure (BP), heart rate (HR), plasma and urinary catecholamine, sodium and potassium, plasma renin and aldosterone levels, and urinary prostaglandin E2 and F2 excretion rates were determined before and up to 4 or 5 h (urine values) after intravenous injection of placebo (20 ml 0.9% NaCl), tiapamil 1 mg/kg body weight, nisoldipine 6 micrograms/kg, or nifedipine 15 micrograms/kg. The four studies were performed at weekly intervals according to Latin square design. All three calcium channel blockers significantly (p less than 0.05 or lower) lowered BP and distinctly increased sodium excretion in hypertensive patients, but had only little influence on these parameters in normal subjects. HR was increased in both groups. Changes in BP and HR were maximal at 5 min and largely dissipated 3 h after drug injection. Effects on BP and HR, as well as concomitant mild increases in plasma norepinephrine and renin levels that occurred in both groups, tended to be more pronounced (about double) following nisoldipine than following tiapamil or nifedipine at the dosages given. Plasma aldosterone, epinephrine levels, and prostaglandin excretion rates were not consistently modified. These findings demonstrate that tiapamil and nisoldipine possess distinct antihypertensive properties in humans. Different chronotropic and renin-activating effects of different calcium channel blockers may be determined, at least in part, by a different influence on sympathetic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Prognostic factors in lymph node metastases of prostatic cancer patients: the size of the metastases but not extranodal extension independently predicts survival

AIMS: To analyse tumour characteristics and the prognostic significance of prostatic cancers with extranodal extension of lymph node metastases (ENE) in 102 node-positive, hormone treatment-naive patients undergoing radical prostatectomy and extended lymphadenectomy. METHODS AND RESULTS: The median number of nodes examined per patient was 21 (range 9-68), and the median follow-up time was 92 months (range 12-191). ENE was observed in 71 patients (70%). They had significantly more, larger and less differentiated nodal metastases, paralleled by significantly larger primary tumours at more advanced stages and with higher Gleason scores than patients without ENE. ENE defined a subgroup with significantly decreased biochemical recurrence-free (P = 0.038) and overall survival (P = 0.037). In multivariate analyses the diameter of the largest metastasis and Gleason score of the primary tumour were independent predictors of survival. CONCLUSIONS: ENE in prostatic cancer is an indicator lesion for advanced/aggressive tumours with poor outcome. However, the strong correlation with larger metastases suggests that ENE may result from their size, which was the only independent risk factor in the metastasizing component. Consequently, histopathological reports should specify the true indicator of poor survival in the lymphadenectomy specimens, which is the size of the largest metastasis in each patient.

Thrombophilic risk factors in patients with cranial and spinal dural arteriovenous fistulae

OBJECTIVE: Numerous studies have reported the technical aspects and results of surgical and/or endovascular treatment of cranial dural arteriovenous fistulae (cDAVF) and spinal dural arteriovenous fistulae (sDAVF). Only a few of them have addressed the question of thrombophilic conditions, which may be relevant as pathogenetic factors or can increase the risk for venous thromboembolic events. Therefore, the objective of this study is to compare thrombophilic risk factors in patients with cDAVF and sDAVF with no history of trauma. METHODS: A total of 43 patients (25 with cDAVF and 18 with sDAVF) were included in this study. Blood samples were analyzed for G20210A mutation of the prothrombin gene and factor V Leiden mutation. In all patients, prothrombin time, international normalized ratio, fibrinogen, antithrombin, protein C and S activity, von Willebrand factor antigen, ristocetin cofactor activity, D-dimer, coagulation factor VIII activity, and tissue factor pathway inhibitor were determined. Screening was performed for the occurrence of lupus antiphospholipid and cardiolipin antibodies. RESULTS: The prevalence of G20210A mutation of the prothrombin gene was significantly higher in patients with cDAVF (n = 6) compared with patients with sDAVF (n = 0; P < 0.05, Fisher's exact test). A factor V Leiden mutation was found in 3 patients with sDAVF and in 1 patient with cDAVF (P = 0.29, Fisher's exact test). No significant difference was found for other parameters, except for fibrinogen, but decreased protein C activity was more frequent in patients with cDAVF compared with patients with sDAVF (4 versus 1). Decreased protein S activity was encountered in 3 patients (2 with sDAVF and 1 with cDAVF). Cardiolipin antibodies were found in 2 patients with cDAVF but in none with sDAVF, whereas only 1 patient with sDAVF had lupus antiphospholipid antibodies. CONCLUSION: In both groups of patients with dural arteriovenous fistulae, genetic thrombophilic abnormalities occurred in a higher percentage than in the general population. The differences of the genetic abnormalities may be involved in different pathophysiological mechanism(s) in the development of these distinct neurovascular entities.

Exploration of the role of serum factors in maintaining bone mass during hibernation in black bears

Disuse osteoporosis is a condition in which reduced mechanical loading (e.g. bed-rest, immobilization, or paralysis) results in unbalanced bone turnover. The American black bear is a unique, naturally occurring model for the prevention of disuse osteoporosis. Bears remain mostly inactive for up to half a year of hibernation annually, yet they do not lose bone mechanical strength or structural properties throughout hibernation. The long-term goal of this study is to determine the biological mechanism through which bears maintain bone during hibernation. This mechanism could pinpoint new signaling pathway targets for the development of drugs for osteoporosis prevention. In this study, bone specific alkaline phosphatase (BSALP), a marker of osteoblast activity, and tartrate resistant acid phosphatase (TRACP), a marker of osteoclast number, were quantified in the serum of hibernating and active black bears. BSALP and TRACP decreased during hibernation, suggesting a balanced reduction in bone turnover. This decrease in BSALP and TRACP were correlated positively to serum adiponectin and inversely to serum neuropeptide Y, suggesting a possible role of these hormones in suppressing bone turnover during hibernation. Osteocalcin (OCN) and undercarboxylated OCN increased dramatically in the serum of hibernating bears. These increases were inversely correlated with adiponectin, glucose, and serotonin, suggesting that OCN may have a unique role in energy homeostasis during hibernation. Finally, MC3T3-E1 osteoblasts were cultured in the serum from active and hibernating bears, and seasonal cell responses were quantified. Cells cultured in serum from hibernating bears had a reduced caspase-3/7 response, and more living cells, after apoptotic threat. The caspase-3/7 response was positively correlated to serum adiponectin and to gene expression of OCN and Runx2, suggesting that reduced caspase-3/7 activity may be related to the reduced differentiation potential of osteoblasts in hibernation serum, and that adiponectin is a potential effector hormone. In summary, the activities of osteoblasts and osteoclasts are reduced during hibernation in bears. This reduced turnover is due, in part, to hormonal control. Further study of potential effectors adiponectin and neuropeptide Y may provide insight into the biological mechanism through which bears maintain bone throughout hibernation.